Among the myosin superfamily of motor proteins, myosin II is the sole filament forming isoform. Myosin IIs are hexamers containing two identical heavy chains and two pairs of light chains, the essential light chain and the regulatory light chain (RLC). Phosphorylation of the RLC by myosin light chain kinase is a general phenomenon in myosin IIs but the effect is variable depending on the source. The actin activated ATPase of both smooth and non-muscle myosin is regulated primarily by RLC phosphorylation. Vertebrate striated muscle myosins (cardiac and skeletal) also undergo RLC phosphorylation but the effect is only to modulate the ATPase activity which is regulated primarily by the troponin-tropomyosin complex on the thin filament. ATPase activity in many invertebrate striated muscle myosins is also controlled via myosin, not through light chain phosphorylation, but by Ca2+ binding to myosin at the interface between ELC and RLC. A common feature of myosin II regulation is the cooperative interaction between both myosin heads; myosin fragments that contain only one head are not regulated and are always "on" with respect to their actin activated ATPase activity. We have recently obtained 2-D arrays of smooth muscle HMM, a two-headed, regulated fragment of myosin II, in both the dephosphorylated "off" and phosphorylated "on" states. These arrays are suitable for 3-imaging by electron crystallography and allow us to visualize for the first time, the head to head interactions that give rise to ATPase inhibition. The 2-D crystallization of HMM from myosin II facilitates study of regulation in a way not previously accessible. This application proposes to extend our observations on smooth muscle myosin regulation to other isoforms of myosin II, such as scallop striated, non-muscle and vertebrate striated muscle myosins in order to determine the generality of the structural interactions.